Endothelin is a potent vasoconstrictor peptide of endothelial origin (Yanagisawa, M. et al., Nature 1988, 332: 411-415), and may represent a family of vasoconstrictors. Three distinct endothelin isopeptides are known (termed herein as Et-1, Et-2, Et-3) all of which cause vasoconstriction in a number of vascular beds. Et-1 is a 21-residue peptide with two disulfide bonds and shows extensive homology with the two other known forms of the peptide (Hirata, Y., et al., Biochem. Biophys Res. Commun., 1984, 160; 228-34).
A structural model based on the endothelin amino acid sequence has been proposed. The molecule is amphipathic consisting of a rigid, disulfide bonded, hydrophilic amino-terminal half of the molecule with two turns, and has extended hydrophobic sheet structure comprising the C-terminal half of the molecule (Spinella, M. J., et al., Peptide Res., 1989 2, 286-91). The extended structure may be stabilized by intermolecular hydrogen bonding, leading to dimers or higher order aggregates in solution and is hydrophobic enough to partition into an organic solvent such as ether, as long as the molecule can keep its hydrophilic amino terminus in an aqueous phase. Several different types of microcrystals of Et-1 have been formed at an aqueous/organic boundary in a two-phase system but precise tertiary structure of endothelin via X-ray crystallography has not yet been established.
In addition, certain peptide toxins found in the venom of the asp, Atractaspis engaddensis, termed sarafotoxins S, are known which have endothelin activity causing severe coronary vasospasm in snake bite victims. The sarafotoxins display significant structural and functional homology to the endothelins. The amino acid sequence of the endothelins and sarafotoxins S are shown hereinbelow in Table 1.
TABLE 1 __________________________________________________________________________ Amino Acid Sequence.sup.1 Of Naturally Occurring Endothelin Active Peptides Peptide Designation.sup.2 1 5 10 15 20 __________________________________________________________________________ Et-1 Cys Ser Cys Ser Ser Leu Met Asp Lys Glu Cys Val Tyr Phe Cys His Leu Asp Ile Ile Trp Et-2 Cys Ser Cys Ser Ser Trp Leu Asp Lys Glu Cys Val Tyr Phe Cys His Leu Asp Ile Ile Trp Et-3 Cys Thr Cys Phe Thr Tyr Lys Asp Lys Glu Cys Val Tyr Tyr Cys His Leu Asp Ile Ile Trp SB Cys Ser Cys Lys Asp Met Thr Asp Lys Glu Cys Leu Tyr Phe Cys His Gln Asp Val Ile Trp SA Cys Ser Cys Lys Asp Met Thr Asp Lys Glu Cys Leu Asn Phe Cys His Gln Asp Val Ile Trp SC Cys Ser Cys Lys Asp Met Thr Asp Glu Glu Cys Leu Asn Phe Cys His Gln Asp Val Ile Trp __________________________________________________________________________ .sup.1 Amino acid positions are numbered starting at the Nterminal cysteine moiety. .sup.2 Et1, Et2, Et3 denote Endothelin 1, Endothelin 2 and Endothelin 3 respectively. SB, SA, SC denote sarafotoxin S6b, Sarafotoxin S6al and Sarafotoxin S6c respectively.
The endothelin active polypeptides depicted in Table 1 all contain 21 amino acid residues. All of them possess a cysteine residue at positions 1, 3, 11 and 15 from the N-terminus. In fact the native peptide depicted in Table 1 is folded so that the cysteines at positions 1 and 15 form a disulfide bond and the cysteines at positions 3 and 11 form another disulfide bond. For example, the amino acid sequence of Et-1 is depicted hereinbelow. ##STR2##
The Et-1 depicted hereinabove specifically shows that the cysteines at positions 1 and 15 form a bond and the cysteines at positions 3 and 11 form another bond. A closer examination of Et-1 clearly reveals that the linkages are disulfide bonds: ##STR3##
In the above formula, the chemical formula for the cysteine residues at positions 1, 3, 11 and 15 are specifically drawn. As clearly shown, the atoms connecting the cysteines at positions 1 and 15 and the cysteines at position 3 and 11 are CH.sub.2 --S--S--CH.sub.2. The atoms linking the amino acids at position 1 and 15 together with the amino residues between these two positions inclusively, constitute an "outer bridge or loop", while those atoms linking the amino acids at position 3 and 11 together with the amino acid residues between positions 3 and 11 inclusively, constitute the "inner bridge or loop".
Before proceeding further, the terminology used will be briefly explained. Peptides are identified by amino acid sequence using established abbreviations. For example, as used herein, in the case of the commonly occurring amino acids, "Gly" stands for glycine, "Leu" stands for Leucine, "Cys" stands for cysteine, etc. Except for glycine, the amino acids depicted in the above table are considered to exist as stereoisomers in the L-configuration. Et-1, Et-2, Et-3, SA, SB, SC designate known endothelin-active polypeptides whose sequences are shown in Table 1. Amino acid moieties in polypeptides are numbered sequentially starting at the N-terminus. Positions of amino acids within the polypeptides are represented by superscripts adjacent to the amino acid designations. For example, position 1 in the naturally occurring endothelin-active polypeptides is occupied by cysteine in all cases and can be represented by "Cys.sup.1 ". Phenylalanine at position 4 in Et-3 is represented as "Phe.sup.4 " Leucine at position 12 of SB as "Leu.sup.12 " trytophan at position 6 of Et-2 as "Trp.sup.6 " and so on. Truncated peptides of the endothelin-active series will be designated with reference to the sequences in Table 1. Thus "Et-1.sup.1-10 " denotes a decapeptide with the amino acid sequence shown for the first ten positions in Et-1 (See Table 1), "Et-3.sup.15-20 ", a hexapeptide with amino acid sequence shown for positions 15-20 for Et-3; and "SB.sup.1-20 ", a peptide with sequence shown for the first 20 positions of SB.
Analogs created by substitution of amino acids or other chemical moieties for the known amino acids of endothelin-active peptides are designated according to position and the amino acid substitution. Thus, an endothelin analog of Et-1 with alanine substituted for cysteine at positions 1 and 15 is designated [Ala.sup.1,15 ]Et-1, and an analog of Et-2 with phenylalanine at position 21 is designated "[Phe.sup.21 ]Et-2".
The positions of disulfide bridges between cysteine moieties are designated by connecting their position numbers with a dash. Thus, naturally occurring endothelin-1 containing two disulfide bridges may be depicted as "[Cys.sup.1-15, Cys.sup.3-11 ]Et-1" (when referring to the disulfide bridge structure) On the other hand "[Cys.sup.1,15, Cys.sup.3-11 ]Et-1" denotes the same polypeptide with reduced cysteine at positions 1 and 15 (no disulfide bridge between the positions).
The positions of the bridging structure replacements for the disulfide bridges between cysteine moieties of the native endothelins is denoted connecting the replacement amino acids designations/positions with a line ##STR4## For example, ##STR5## denotes that the Cys.sup.1-15 disulfide constituting the outer loop bridging structure of Et-1 has Cys replaced with Dpr (diaminopropionic acid moiety) at position 1 with Asp at position 15, and that the two amino acid moieties participate in a bridging structure, for example, through an amide bond.
Analogs of the polypeptides in Table 1 wherein one or more amino acid substitutions, additions or deletions have been made are known. For example, [Ala.sup.3,11 ]Et-1 shows endothelin activity (Randall, et al., Br. J. Pharmacol, 1984, 98, 685-699). [Homoserine.sup.6 ]SB shows endothelin activity (Kitazuni, FEBS Letters, 1990, 260, 269-72). [Ala.sup.4 ]Et-1, [Ala.sup.5 ]Et-1, [Gly.sup.6 ]Et-1, [Met(O).sup.7 ]Et-1, [Asn.sup.8 ]Et-1, [Leu.sup.9 ]Et-1, [Phe.sup.13 ]Et-1, [Tyr.sup.21 ]Et-1, and [Phe.sup.21 ]Et-1 show appreciable endothelin activity (Nakajima, et al., Biochem Biophys Res Commu., 1989, 163, 424-9). [By definition, [met] [O] refers to an oxidized methionine e.g. the side group is ##STR6## Yet other types of analogs of the polypeptides shown in Table 1 have been shown to have endothelin activity, for example Et-1 in which the Glu.sup.10 residue has been anisylated (Hiley, et al., Br. J. Pharmacol, 1990, 101, 319-24) and Lys-Arg-Et-1 (Et-1 extended through the addition of lysylarginine to the N-terminus; see Nakajima, op. cit.). Endothelin precursors, i.e., polypeptides containing more than 21 amino acids, which when cleaved chemically or enzymatically produce an endothelin active polypeptide, have been identified. For example, endothelin precursors with 38 (human) or 39 (porcine) amino acids have been identified (so called "Big Et-1") which are subsequently processed in vivo by an endothelin cleaving enzyme to produce mature Et-1 (Yanigasawa, et al., Biochem. Pharmacol. 1,989, 38, 1877-83); Yanigasawa, et al., Nature, 1988, 332, 411-15). It is contemplated that several such polypeptide precursors exist or otherwise could be synthesized by art known methods which are processable in vivo to form endothelin-active polypeptides.
The mode of action of the endothelins and sarafotoxins in eliciting vasoconstriction is still a matter of intense inquiry. The three distinct endothelins cause vasoconstriction in a number of vascular beds with an apparent potency order of: Et-2&gt;Et-1&gt;Et-3 (Anggard, et al. Blood. Vessels 1990, 29 269-81). Two distinct endothelin receptors have been cloned, one of which appears to be specific for Et-1 (Arai., et al. Nature, 1990, 348, 730-2) while the other interacts with all three Et polypeptides (Sakurai et al Nature 1990, 348 732-4).
Competitive binding studies have suggested multiple classes of receptors with varying affinities for the different endothelins anti that the distribution of the receptor subtypes is tissue specific (Simonson, et al FASEB J., 1990, 4, 2989-3000); Martin. et al. Biochem. Biophys. Res Commun. 1989, 162, 130-7); Kloog, et al. FEBS Letters, 1989, 253, 199-202. Thus, it should be appreciated that more than one type of endothelin-active polypeptide exists and that more may be discovered with widely different levels of activity in different tissues. In like manner, the endothelin antagonists of the present invention may be tissue specific since they are structural analogs of the endothelin-active polypeptides.
It is important to note that the known endothelins, whether naturally occurring or synthesized, have a disulfide bridge and more specifically a CH.sub.2 --S--S--CH.sub.2 group in the outer bridge. The cysteine at positions 1 and 15 and the outer bridge is believed to be necessary for the polypeptides in Table 1 to possess endothelin activity.
A recent report by Fabregat, et al. (J. Cellular Physiol 1990 145:88-94) describes a substance P based peptide analog ([D-Arg.sup.1, D-Phe.sup.5, D-Trp.sup.7,9, Leu.sup.11 ] substance P) that blocks certain endothelin responses and is able to inhibit .sup.125 I-labelled Et-1 binding to an Et-1 receptor in a competitive and dose dependent manner. This peptide is an undecapeptide containing no cysteine residues. Neither is it related in structure to the known endothelin-active peptides or to the novel endothelin antagonists and precursors which are the subject of the present invention.